Application of SRT1720 in promoting in vitro maturation of sheep oocytes and improving embryonic development

Abstract

This invention relates to the field of in vitro embryo production technology for livestock, and particularly to the application of SRT1720 in promoting in vitro maturation of sheep oocytes and improving embryo development. The SIRT1-specific agonist SRT1720 is used to promote the in vitro maturation rate of sheep oocytes, or in the preparation of pharmaceuticals or health products that promote the in vitro maturation rate of sheep oocytes. This invention adds SRT1720 to conventional oocyte in vitro maturation culture medium, which can significantly improve the maturation rate of sheep oocytes, reduce the proportion of spindle morphological abnormalities and chromosome misalignment, improve mitochondrial function and reduce mitochondrial ROS accumulation, thereby significantly improving the cleavage and blastocyst development capabilities of in vitro fertilized embryos, and effectively improving the quality of in vitro oocyte maturation and embryo developmental potential.

Description

Technical Field

[0001] This invention relates to the field of in vitro embryo production technology for livestock, and particularly to the application of SRT1720 in promoting in vitro maturation of sheep oocytes and improving embryo development. Background Technology

[0002] Animal embryo engineering technology is a core means to achieve rapid breeding of superior breeds, somatic cell cloning, and transgenic animal production. Among these technologies, large-scale sheep embryo production has significant application value for genetic improvement in animal husbandry. Currently, the conventional method of obtaining in vivo embryos through superovulation suffers from significant drawbacks, including large individual differences in donors, low embryo yield, high labor and material costs, and difficulty in providing stable, large-scale supply. These drawbacks severely restrict the large-scale and industrial application of sheep embryo engineering technology. In vitro maturation (IVM) of oocytes is the core initiating step in in vitro embryo production, directly determining the synchronous nucleocytoplasmic maturation of oocytes, subsequent fertilization, and embryonic developmental potential. However, existing in vitro maturation systems differ significantly from the in vivo physiological environment, leading to problems such as low maturation rates, asynchronous nucleocytoplasmic maturation, mitochondrial dysfunction, abnormal spindle assembly, and chromosome misalignment in in vitro matured oocytes. Ultimately, this results in oocyte quality and developmental potential that are significantly lower than those of in vivo matured oocytes, becoming a key technological bottleneck limiting the efficiency of sheep in vitro embryo production.

[0003] SIRT1, a core member of the Sirtuins family, is a key deacetylase regulating cellular energy metabolism, mitochondrial homeostasis, oxidative stress levels, and meiosis, playing a crucial regulatory role in oocyte maturation. SRT1720, a SIRT1-specific agonist, can target and activate the SIRT1 signaling pathway, improving mitochondrial biosynthesis and functional homeostasis, maintaining cellular redox balance, and thus ensuring normal meiosis and stable spindle and chromosome structures in oocytes. Currently, in traditional sheep oocyte in vitro maturation systems, there are no reports of using SRT1720 as a highly effective additive to improve oocyte maturation quality, and maturation strategies that target and improve mitochondrial function and enhance oocyte developmental potential are still lacking.

[0004] Therefore, the present invention aims to provide a culture medium additive for promoting in vitro maturation of sheep oocytes. The additive is SRT1720. By adding SRT1720 to the conventional oocyte in vitro maturation culture medium, the maturation rate and quality of sheep oocytes can be significantly improved, mitochondrial function, spindle morphology and chromosome arrangement can be improved, and the developmental potential of subsequent fertilized embryos can be enhanced. This effectively solves the technical problem of low efficiency in the existing in vitro maturation system and provides stable and efficient technical support for the large-scale application of sheep embryo engineering. Summary of the Invention

[0005] The purpose of this invention is to provide the application of SRT1720 in promoting in vitro maturation of sheep oocytes and improving embryo development, effectively solving the technical problem of low efficiency in existing in vitro oocyte maturation systems, and providing stable and efficient technical support for the large-scale application of sheep embryo engineering.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of SRT1720, a SIRT1-specific agonist, in promoting the in vitro maturation rate of sheep oocytes, or in the preparation of drugs or health products that promote the in vitro maturation rate of sheep oocytes.

[0008] Furthermore, the concentration of the SIRT1 specific agonist SRT1720 is 5-20 μmol / L.

[0009] Furthermore, the concentration of the SIRT1 specific agonist SRT1720 is 10 μmol / L.

[0010] Any of the following applications of the SIRT1-specific agonist SRT1720:

[0011] 1) Reduce the proportion of abnormal spindle morphology in sheep oocytes, or prepare drugs or health products that reduce the proportion of abnormal spindle morphology in sheep oocytes;

[0012] 2) Reduce the proportion of abnormal chromosome arrangement in sheep oocytes, or prepare drugs or health products that reduce the proportion of abnormal chromosome arrangement in sheep oocytes;

[0013] 3) Improve the quality of sheep oocyte mitochondria, or prepare drugs or health products that improve the quality of sheep oocyte mitochondria;

[0014] 4) Improve the quality of sheep oocyte maturation, or prepare drugs or health products that improve the quality of sheep oocyte maturation.

[0015] Furthermore, the improvement of sheep oocyte mitochondrial quality includes any of the following applications:

[0016] 1) Increase the number of mitochondria and / or enhance mitochondrial membrane potential;

[0017] 2) Reduce ROS levels;

[0018] 3) Increase ATP levels.

[0019] Furthermore, the improvement of sheep oocyte maturation quality includes increasing sheep cleavage rate and blastocyst rate.

[0020] Furthermore, the concentration of the SIRT1 specific agonist SRT1720 is 5-20 μmol / L.

[0021] Furthermore, the concentration of the SIRT1 specific agonist SRT1720 is 10 μmol / L.

[0022] This invention has at least the following beneficial effects:

[0023] This invention aims to address the technical bottlenecks of poor quality and low embryo development efficiency in existing sheep oocyte in vitro maturation culture media. It provides an additive for promoting sheep oocyte in vitro maturation, namely SRT1720, used at a concentration of 5-20 μM. Adding SRT1720 to conventional oocyte in vitro maturation culture media significantly improves sheep oocyte maturation rate, reduces the proportion of spindle morphological abnormalities and chromosome misalignment, improves mitochondrial function and reduces mitochondrial ROS accumulation, thereby significantly enhancing the cleavage and blastocyst development capabilities of in vitro fertilized embryos, effectively improving oocyte in vitro maturation quality and embryo development potential. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A schematic diagram illustrating the effect of adding different concentrations of SRT1720 to the in vitro maturation medium of oocytes on the maturation rate of sheep oocytes.

[0026] Figure 2 A schematic diagram showing the effect of adding different concentrations of SRT1720 to the in vitro maturation medium of oocytes on spindle morphology and chromosome arrangement.

[0027] Figure 3 A schematic diagram illustrating the effect of adding different concentrations of SRT1720 to the in vitro maturation medium of oocytes on the mitochondrial abundance of sheep oocytes.

[0028] Figure 4 A schematic diagram showing the effect of adding different concentrations of SRT1720 to the in vitro maturation medium of oocytes on the mitochondrial ROS content of sheep oocytes.

[0029] Figure 5 A schematic diagram illustrating the effect of adding different concentrations of SRT1720 to the in vitro maturation medium of oocytes on the mitochondrial ATP levels of sheep oocytes. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] The ovaries used in the experiment were collected from Chongqing Jinhang Halal Slaughterhouse. TCM199 and fetal bovine serum used in this invention were purchased from Gibco, and other reagents, unless otherwise specified, were purchased from Sigma-Aldrich.

[0032] I. Experimental Methods

[0033] 1. Culture medium preparation

[0034] In vitro maturation medium for oocytes: M199 culture medium was supplemented with 10% (v / v) fetal bovine serum (FBS), 1% (v / v) insulin-transferrin (ITS), 0.1 IU / mL follicle-stimulating hormone (FSH), 2 µg / mL β-estradiol, 0.2 mM sodium pyruvate, 10 ng / mL EGF, 100 IU / mL penicillin, and 100 mg / mL streptomycin. After filtration and aliquoting, the medium was stored at 4°C for later use (see Table 1).

[0035] Table 1. Formula for in vitro maturation solution of sheep oocytes

[0036] Fetal bovine serum (FBS) 10% (v / v) Insulin transferrin (ITS) 1% (v / v) Follicle-stimulating hormone (FSH) 0.1 IU / mL β-estradiol 2 µg / mL Sodium pyruvate 0.2 mM Epidermal growth factor (EGF) 10 ng / mL penicillin 100 IU / mL Streptomycin 100 mg / mL

[0037] Egg washing solution: M199 culture medium with 5% (v / v) FBS and 0.1% (v / v) PVA added.

[0038] In vitro fertilization solution (capacitation solution): Add 50 µg / mL heparin, 2.5 mM caffeine, 6 mg / mL BSA, 0.2 mM sodium pyruvate, 100 IU / mL penicillin, and 100 mg / mL streptomycin to the in vitro fertilization (IVF) base solution, filter and aliquot, and store at 4°C for later use (see Table 2).

[0039] Table 2 In Vitro Fertilization Fluid Formulation Table

[0040] <![CDATA[CaCl2]]> 222.2000 mg / L <![CDATA[MgCl2.6H2O]]> 101.6500 mg / L KCl 238.4000 mg / L <![CDATA[NaHCO3]]> 2100.0000mg / L NaCl 6669.0000 mg / L <![CDATA[C3H5NaO3]]> 1.4160 mg / L <![CDATA[NaH2PO4.H2O]]> 55.2000 mg / L heparin 50mg / L caffeine 2.5 mM Bovine serum albumin (BSA) 6 g / L Sodium pyruvate 0.2 mM penicillin 100 IU / mL Streptomycin 100 mg / mL

[0041] The mSOFaa embryo culture medium is shown in Table 3:

[0042] NaCl 106 KCl 7.2 <![CDATA[NaHCO3]]> 25 <![CDATA[KH2PO4]]> 1.2 Sodium lactate 6.6 <![CDATA[CaCl2.2H2O]]> 1.7 <![CDATA[MgCl2.6H2O]]> 0.5 Sodium pyruvate 0.3 glucose 1.5 BSA 8 mg / mL MEM essential amino acids 2% MEM non-essential amino acids 1% glutamic acid 1 ITS 0.50%

[0043] Table 3. mSOFaa culture medium formulation table

[0044] 2. In vitro maturation culture of oocytes

[0045] Follicular fluid containing cumulus-oocyte complexes (COCs) was collected from follicles with a diameter of 2–8 mm using a disposable syringe with an 8-gauge needle. The follicular fluid was diluted with oocyte washing solution, and COCs containing at least three layers of cumulus cells were selected under an optical microscope. After washing the COCs three times in the oocyte washing solution, they were rinsed once in maturation culture medium. Then, groups of 50 COCs were placed in concave glass dishes containing 750 μL of maturation culture medium and incubated for 24 h in an incubator at 38.5°C, saturated humidity, and 5% CO2.

[0046] 3. In vitro fertilization of oocytes and embryo culture

[0047] Sperm capacitation: Place one frozen capillary tube stored in liquid nitrogen in a 37°C water bath and thaw for 1 min. Transfer the sheep semen into a 5 mL test tube containing 2 mL of sperm capacitation solution (equilibrated for at least 2 h beforehand). Tilt the test tube at a 45-degree angle in a 37°C incubator for 30 min to allow the sperm to float. After the sperm floats, aspirate approximately 1.5 mL of the upper and middle layers (avoid aspirating turbid substrate). Centrifuge the liquid containing the floated sperm (1000 rpm) for 10 min, discard the supernatant, and retain 200–500 μL of liquid at the bottom of the test tube.

[0048] In vitro fertilization of oocytes: Mature oocytes were selected under a microscope and washed three times in a balanced capacitation solution. A group of 30 mature oocytes was placed in a 50 μL droplet of fertilization solution and covered with mineral oil. 50 μL of capacitation sperm was added to each droplet, and the mixture was then incubated at 37°C with 5% CO2 for 8–10 h.

[0049] Embryo culture: The oocytes that are presumed to be fertilized are washed three times in balanced mSOFaa medium. Then, 20 oocytes are placed in a group in 100 μL of mSOFaa droplets and covered with mineral oil. The cells are cultured at 38.5℃, 5% CO2, and saturated humidity for 7 days, with 70% of the medium changed every 3 days.

[0050] 4. Statistics on oocyte maturation rate

[0051] After 22-24 hours of in vitro maturation culture, hyaluronidase is added to a four-well plate, and the cells are pipetted 40-60 times until the cumulus cells detach. Oocytes with uniform cytoplasm and extrusion of the first polar body under a stereomicroscope are considered mature.

[0052] 5. Detection of mitochondrial ROS in oocytes

[0053] The ROS content in oocyte mitochondria was detected using mitochondrial superoxide fluorescent dye. Oocytes were incubated in M199 medium containing 10 μM MitoSO™ Red (Beyotime) at 37°C in the dark for 30 min. After washing three times with M199 medium containing 0.1% (w / v) BSA, the oocytes were observed at a wavelength of 460 nm using a fluorescence microscope. Finally, the fluorescence intensity of the oocytes was analyzed using Image-Pro Plus 6.0 software.

[0054] 6. Spindle fibers and DNA staining

[0055] Oocytes were fixed with 4% paraformaldehyde at room temperature for 30 minutes, followed by washing three times with PBS. Next, they were permeabilized with permeabilization buffer at room temperature for 30 minutes, washed three times again with PBS, and then transferred to blocking buffer for 2 hours at room temperature. Afterwards, the cells were incubated overnight at 4°C with primary antibody (α-Tubulin, dilution 1:200), washed three times with PBS, and then incubated with secondary antibody at room temperature in the dark for 2 hours. Finally, the cells were washed three times with PBS, mounted with a DAPI-containing anti-fluorescence quenching mounting medium, and images were acquired using a laser confocal microscope.

[0056] 7. Mitochondrial staining

[0057] Oocytes were incubated in M199 containing 100 nM Mito-Tracker Green (Beyond) at 37 °C in the dark for 30 min. After washing with M199 containing 0.1% BSA, the distribution of mitochondria in the oocytes was observed under a confocal microscope.

[0058] 8. Statistical Analysis

[0059] Statistical analysis was performed using GraphPad Prism 9.0 software. Unpaired two-tailed t-tests were used for comparisons between two independent samples; one-way ANOVA combined with Tukey's post-hoc test was used for comparisons between more than two groups. Normally distributed data are expressed as mean ± standard deviation (mean ± SD), unless otherwise specified. A p-value < 0.05 was considered statistically significant.

[0060] II. Experimental Results and Analysis

[0061] 1. Adding SRT1720 to the maturation solution promotes in vitro maturation of sheep oocytes.

[0062] Compared with the control group, the addition of 5, 10, and 20 μM SRT1720 to the in vitro maturation culture medium significantly increased the maturation rate of sheep oocytes (P < 0.05), with 10 μM (i.e., 10 μmol / L) SRT1720 showing the best effect and the most significant increase in the in vitro maturation rate of oocytes (P < 0.001). Figure 1 ).

[0063] 2. Adding SRT1720 to the maturation solution improves spindle morphology and chromosome alignment in sheep oocytes.

[0064] Compared with the control group, the addition of 10 and 20 μM SRT1720 to the in vitro maturation culture medium significantly reduced the proportion of abnormal spindle structures in sheep oocytes (normal spindles are barrel-shaped), with 10 μM SRT1720 showing the best effect (P < 0.001). Figure 2 (A and B in the text).

[0065] Meanwhile, the addition of 5, 10, and 20 μM SRT1720 significantly reduced the rate of abnormal chromosome alignment (normal chromosomes were neatly aligned) (P < 0.05), with 10 μM SRT1720 showing the most significant improvement (P < 0.001). Figure 2 (A and C in the text).

[0066] 3. Adding SRT1720 to the maturation solution improves the quality of sheep oocyte mitochondria.

[0067] Compared with the control group, the addition of 5, 10, and 20 μM SRT1720 to the in vitro maturation culture medium significantly increased the mitochondrial fluorescence abundance of sheep oocytes (P < 0.05), with 10 μM SRT1720 showing the best effect and the most significant increase in the in vitro maturation rate of oocytes (P < 0.001). Figure 3 Increased fluorescence intensity typically indicates an increase in the number (quality) of mitochondria or an enhancement of mitochondrial membrane potential, which directly reflects the activity and health of the mitochondrial network. This suggests that SRT1720 promotes mitochondrial biosynthesis or functional enhancement.

[0068] Compared with the control group, the addition of 5, 10, and 20 μM SRT1720 to the in vitro maturation culture medium significantly reduced the mitochondrial ROS level of sheep oocytes (P < 0.05), with 10 μM SRT1720 showing the best effect (P < 0.01). Figure 4 ROS (reactive oxygen species) are byproducts of cellular metabolism (especially in the mitochondrial respiratory chain), and excessive accumulation can lead to oxidative damage. The significantly reduced ROS levels indicate that SRT1720 effectively alleviates oxidative stress in oocytes during in vitro maturation, protecting cell structure and DNA integrity.

[0069] Compared with the control group, the addition of 10 and 20 μM SRT1720 to the in vitro maturation culture medium significantly increased the mitochondrial ATP level of sheep oocytes (P < 0.05), with 10 μM SRT1720 showing the best effect (P < 0.01). Figure 4 ATP (adenosine triphosphate) is the energy currency of cells. A significant increase in ATP levels is the most direct evidence of intact mitochondrial function, indicating that oocytes treated with SRT1720 have higher energy production efficiency and can reserve sufficient energy for subsequent maturation division, fertilization and early embryonic development.

[0070] 4. Adding SRT1720 to the maturation solution improves the quality of sheep oocyte maturation.

[0071] Compared with the control group, the addition of 5, 10, and 20 μM SRT1720 to the in vitro maturation culture medium significantly improved the cleavage rate and blastocyst rate of sheep (P < 0.05), with the addition of 10 μM SRT1720 showing the best effect (Table 4).

[0072] Table 4. Effects of different concentrations of SRT1720 on the maturation quality of sheep oocytes.

[0073] control group 154 <![CDATA[106(68.97±3.18) c ]]> <![CDATA[16(10.39±0.86) d ]]> 5μM SRT1720 152 <![CDATA[119(78.32±1.86) b ]]> <![CDATA[21(13.78±0.48) c ]]> 10μM SRT1720 146 <![CDATA[121(82.85±1.36) a ]]> <![CDATA[27(18.46±1.11) a ]]> 20μM SRT1720 141 <![CDATA[111(78.79±5.59) b ]]> <![CDATA[22(15.62±0.70) b ]]>

[0074] Note: Cleavage rate (%) = Number of cleavages ÷ Number of embryos cultured × 100; Blastocyst rate (%) = Number of blastocysts ÷ Number of embryos cultured × 100. Different lowercase letters (a–d) in the same column indicate significant differences (P<0.05).

[0075] In conclusion, the addition of 5-20 μM SRT1720 to the in vitro maturation culture medium can significantly improve the maturation efficiency and mitochondrial function of sheep oocytes, improve spindle assembly and chromosome arrangement abnormalities, and enhance the in vitro developmental potential of fertilized embryos, showing important application prospects in the field of in vitro animal embryo production.

[0076] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. The application of SRT1720, a SIRT1 specific agonist, in promoting the in vitro maturation rate of sheep oocytes, or in the preparation of drugs or health products that promote the in vitro maturation rate of sheep oocytes.

2. The application according to claim 1, characterized in that, The concentration of the SIRT1-specific agonist SRT1720 is 5-20 μmol / L.

3. The application according to claim 2, characterized in that, The concentration of the SIRT1-specific agonist SRT1720 is 10 μmol / L.

4. Any of the following applications of the SIRT1-specific agonist SRT1720: 1) Reduce the proportion of abnormal spindle morphology in sheep oocytes, or prepare drugs or health products that reduce the proportion of abnormal spindle morphology in sheep oocytes; 2) Reduce the proportion of abnormal chromosome arrangement in sheep oocytes, or prepare drugs or health products that reduce the proportion of abnormal chromosome arrangement in sheep oocytes; 3) Improve the quality of sheep oocyte mitochondria, or prepare drugs or health products that improve the quality of sheep oocyte mitochondria; 4) Improve the quality of sheep oocyte maturation, or prepare drugs or health products that improve the quality of sheep oocyte maturation.

5. The application according to claim 4, characterized in that, The improvement of sheep oocyte mitochondrial quality includes any of the following applications: 1) Increase the number of mitochondria and / or enhance mitochondrial membrane potential; 2) Reduce ROS levels; 3) Increase ATP levels.

6. The application according to claim 4, characterized in that, The improvement of sheep oocyte maturation quality includes increasing sheep cleavage rate and blastocyst rate.

7. The application according to claim 4, characterized in that, The concentration of the SIRT1-specific agonist SRT1720 is 5-20 μmol / L.

8. The application according to claim 4, characterized in that, The concentration of the SIRT1-specific agonist SRT1720 is 10 μmol / L.

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